PROJECT SUMMARY/ABSTRACT. CD4+ T regulatory cells (Treg cells) are a minor population of lymphocytes found throughout the body. Treg cells are primarily characterized by expression of the transcription factor Foxp3 and are essential for preventing autoimmunity throughout life. Because of their ability to suppress the activity of pro-inflammatory cells, and recent success treating human diseases, Treg cells can potentially be effective in treating inflammatory bowel disease (IBD). Some of the obstacles limiting the clinical use of Treg cells include difficulties obtaining a sufficient number of cells, the inability to direct the cellstothesiteofinflammation,andtheinstabilityofsomeTregcellsunderinflammatoryconditions.Thelatter is especially trueof in vitro-differentiated Treg cells (iTreg cells) that canbe generated bystimulation of nave precursors in the presence of transforming growth factor-beta (TGF?), interleukin-2 (IL-2) and retinoic acid. ThisapproachwillallowforthegenerationoflargernumbersofTregcellsthancanbeisolatedandexpanded from peripheral blood. Moreover, such cells can be engineered to express gut antigen-specific receptors that willultimatelyguidethemtotheinflamedintestines.Thismeansthattheonlymajorhurdlethatwouldremainis the well-documented instability of iTreg cells. Finding ways to overcome this limitation could therefore hasten the therapeutic use of iTreg cells. One way to address this is to first determine how Treg cell stability is achieved in nature and apply those mechanisms to the generation of iTreg cells. We have found that in the absence of T cell so-stimulation via the interaction of the inducible T cell co-stimulator (ICOS) and its ligand (ICOSligand,ICOSL),Tregcellsinthelargeintestine-allofwhichdevelopnaturallyinvivo-loseexpression of Foxp3.Even in health, the intestine is consideredto be in a stateof ?physiologic inflammation?owing to the basallevelsofpro-inflammatorymediatorsinducedviainteractionswiththecommensalmicrobiota.Therefore, this project will test the hypothesis that ICOSL-ICOS stimulation is essential for imprinting the phenotypic stabilitythatpromotesFoxp3+Tregcellresilienceinthepresenceofpro-inflammatorysignals.Importantly,we havealsofoundthatiTregcellsdifferentiatedintheabsenceofICOSLrapidlyextinguishexpressionofFoxp3, in stark contrast to their ICOSL-activated counterparts. The first objective of this proposal is to determine the possible consequences of ICOSL deficiency on the fate of in vivo derived Treg cells, providing novel insight into nature?s use of this pathway for promoting sustained expression of Foxp3 in the intestines. We will then test whether inclusion of a robust APC-derived ICOSL signal is an effective means of inducing stable microbiota antigen-specific iTreg cells that can specifically impede ongoing gut inflammation. Ultimately, this studyhasthepotentialtomoveusonestepclosertoeffectiveiTregcell-mediatedimmunotherapyforIBD.